This proposal sets forth an integrated series of chemical and structural studies to elucidate biologically relevant structure-reactivity correlations of the metal binding protein, metallothionein (MT). This protein is a normal constituent of mammalian tissues and it or related structures are found in a wide vartiety of non-mammalian species. It is the principal intracellular binding site for the environmentally important, toxic heavy metal Cd and is thought to provide the means for its detoxification. A central role has also been proposed for MT in the metabolism of the essential metals Zn and Cu. Depending on the physiological status of the organism in which it is found, the protein can contain more than one type of metal and, in fact, is most often found in a mixed-metal state. The overall goal of the proposed research is to rationalize on the basis of in vitro metal exchange reactions how these mixed metal MTs are formed in vivo and to determine the structural and chemical conseqeueces of shared occupancy of the metal-thiolate clusters by different metals. The structural probes to be used are 113Cd and 1H NMR and they will be interactively employed to provide complementary information regarding the environments of the metals at each of the seven binding sites and the protein structure surrounding these sites. The following specific goals will be pursued: (1) to determine structural diffrences that exist between MT isoproteins and between the two metal-thiolate clusters as a function of the metal bound, (2) to characterize the binding of Cd to apoMT, (3) to investigate the kinetics and mechanisms of reactions which produce mixed Cd,ZnMTs, (4) to characterize the products of in vitro reactions which produce mixed Cu,ZnMTs and Cu,CdMTs, and (5) to assess the biological relevance of these reactions by comparing their products to those of native rabbit liver Cu,ZnMT.